Cellular penetration and antisense activity by a phenoxazine-substituted heptanucleotide

One of the major barriers to the development of antisense therapeutics has been their poor bioavailability. Numerous oligonucleotide modifications hav e been synthesized and evaluated for enhanced cellular permeation with limi ted success. Phenoxazine, a tricyclic 2' deoxycytidine analog, was designed to improve stacking interactions between heterocycles of oligonucleotide/R NA hybrids and to enhance cellular uptake. However, the bioactivity and eel lu lar permeation properties of phenoxazine-modified oligonucleotides were unknown. Incorporation of four phenoxazine bases into a previously optimiz ed C-5 propyne pyrimidine modified 7-mer phosphorothioate oligonucleotide t argeting SV40 large T antigen enhanced in vitro binding affinity for its RN A target and redirected RNAse H-mediated cleavage as compared with the I-me r C-5 propynyl phosphorothioate oligonucleotide (S-ON). The phenoxazine/C-5 propynyl U 7-mer S-ON showed dose-dependent, sequence-specific, and target -selective antisense activity following microinjection into cells. Incubati on of the phenoxazine/C-5 propynyl U S-ON with a variety of tissue culture cells, in the absence of any cationic lipid, revealed unaided cellular pene tration, nuclear accumulation, and subsequent antisense activity. The uniqu e permeation properties and gene-specific antisense activity of the 7-mer p henoxazine/C-5 propynyl U S-ON paves the way for developing potent, cost-ef fective, self-permeable antisense therapeutics.